Synthesis of peptides by the solid-phase method. 6. Neurotensin, fragments, and analogues.

Neurotensin (NT) and 24 related compounds, including fragments or analogues modified at the C-terminal end of the parent molecule, have been prepared by the solid-phase method. After purification by cation-exchange chromatography, the compounds were characterized by thin-layer chromatography, amino acid analysis, elemental analysis, and high-pressure liquid chromatography. The stimulating effects of the peptides were evaluated in rat stomach strips, in isolated spontaneously beating atria of guinea pigs, and in the coronaries of perfused rat hearts. The differences between the biological activities of these compounds are discussed.

Use of papain as a model for the structure-based design of cathepsin K inhibitors: crystal structures of two papain-inhibitor complexes demonstrate binding to S'-subsites.

Papain has been used as a surrogate enzyme in a drug design effort to obtain potent and selective inhibitors of cathepsin K, a new member of the papain superfamily of cysteine proteases that is selectively and highly expressed in osteoclasts and is implicated in bone resorption. Here we report the crystal structures of two papain-inhibitor complexes and the rational design of novel cathepsin K inhibitors. Unlike previously known crystal structures of papain-inhibitor complexes, our papain structures show ligand binding extending deep within the S'-subsites. The two inhibitor complexes, carbobenzyloxyleucinyl-leucinyl-leucinal and carbobenzyloxy-L-leucinyl-L-leucinyl methoxymethyl ketone, were refined to 2.2- and 2.5-A resolution with R-factors of 0.190 and 0. 217, respectively. The S'-subsite interactions with the inhibitors are dominated by an aromatic-aromatic stacking and an oxygen-aromatic ring edge interaction. The knowledge of S'-subsite interactions led to a design strategy for an inhibitor spanning both subsites and yielded a novel, symmetric inhibitor selective for cathepsin K. Simultaneous exploitation of both S- and S'-sites provides a general strategy for the design of cysteine protease inhibitors having high specificity to their target enzymes.

Aurosomes produced by sodium chloroaurate.

Aurosomes were produced in the rabbit synovial membrane by injecting sodium chloroaurate. They contained electron-dense, filamentous, rod-like and lamellar profiles studded with particles and granules. These aurosomes are morphologically indistinguishable from aurosomes produced by sulphur-containing soluble gold salts such as sodium aurothiomalate and aurothioglucose. It is therefore concluded that the sulphur-containing portion of these molecules is not responsible for the characteristic morphology of the aurosome as claimed by others. However, electron-probe x-ray analysis of aurosomes produced by sodium chloroaurate showed the presence of gold, phosphorus and sulphur. It is concluded that the sulphur and phosphorus must have been derived from the biological milieu, since it could not have been derived from the injected compound.

Uraniosomes produced in the synovial membrane by uranyl acetate.

Uranyl acetate was injected into the rabbit knee joint. This produced single-membrane-bound presumably lysosmal bodies (called 'uraniosomes') containing electron-dense crystals in Type A and Type B synovial intimal cells, subsynovial macrophages and lipocytes. Uranium deposits were also seen in the extracellular matrix. All uraniosomes and extracellular deposits analysed by electron-probe X-ray analysis were found to contain uranium, potassium and phosphorus. Traces of calcium and sulphur were also found in some of the uraniosomes and extracellular uranium deposits.

Carrageenan-induced intestinal injury in the rat--a model for inflammatory bowel disease.

The cause of inflammatory bowel disease (IBD) remains unknown. In this report, an attempt is made to produce a suitable animal model for studying the pathobiology of IBD, especially its pathogenesis. Sprague-Dawley rats were divided into four groups of six (A, B, C, D). The experimental design involved prior parenteral sensitization of groups A and B by a 1.5 percent solution of lambda degraded carrageenan followed by oral administration of the same solution for 30 days to groups A and C. The animals were then sacrificed, and the small intestine was evaluated for injury. Oral carrageenan caused significant intestinal injury as evidenced by ulceration, abnormal villous pattern, degree and extent of inflammation [p = 0.0001 for groups (A + C) versus (B + D)]. Prior sensitization aggravated the effects of oral carrageenan. Overall, the inflammation produced was reminiscent of human IBD in that there was pin-point ulceration, focality of lesions and lymphoid hyperplasia with microgranulomas. It was concluded that this carrageenan model may prove to be particularly useful for studying the pathobiology of human IBD.

Carrageenan-induced intestinal injury: possible role of oxygen free radicals.

There is a growing body of evidence that implicates oxygen free radicals in a wide variety of inflammatory conditions in various body systems including the gastrointestinal tract. The purpose of this study was to ascertain whether or not oxy-radicals play a role in carrageenan-mediated intestinal injury. Allopurinol, superoxide dismutase-polyethylene glycol, and dimethyl sulfoxide, respectively, were administered to the carrageenan rat model for 30 to 32 days. Collectively, all three drugs attenuated the carrageenan-mediated injury as shown by four indices of intestinal damage: ulceration (p = 0.0007); abnormal villous pattern (p = 0.0002); degree of inflammation (p = 0.0001); and extent of inflammation (p = 0.0025). Dimethyl sulfoxide appeared to be the least efficacious of the three drugs. The results suggest that oxygen free radicals play a role in carrageenan-mediated intestinal injury, and that one of the sources of these oxy-radicals may be the intestinal macrophage.

Experimental methods of repairing injured menisci.

A longitudinal incision resembling a bucket-handle tear was made in the menisci of 8 rabbits, 6 dogs, 11 pigs and 12 sheep. In some of the animals of each species the cut was repaired by suturing, and in others it was not. Gross inspection, as well as examination by light and electron microscopy, showed that no healing had occurred after six months in the sutured or the unsutured wounds and that the meniscus was incapable of significant intrinsic repair. In a second experiment longitudinal, transverse and T-shaped cuts were made in the menisci of 12 sheep, and a flap of synovium was sutured into the wound. Three months later there was clear evidence of healing by the formation of cartilaginous tissue. Examination by light and electron microscopy showed that the newly formed repair tissue, possibly derived by metaplasia from the synovium, had a morphology intermediate between hyaline cartilage and fibrocartilage. Synovial implantation may therefore be considered as an alternative to meniscectomy in the management of the torn meniscus.

Electron probe X-ray analysis of an intraocular foreign body.

We describe a simple and rapid method of electron probe x-ray analysis on a foreign body removed from the eye. We demonstrated the presence of copper in an intraocular foreign body which has originated from a blank 0.22 calibre cartridge. Sodium, potassium, calcium, phosphorus, sulpher and chlorine were also detected. It seems likely that these elements were derived from the biological milieu in which the intraocular foreign body had rested for some 2 years and 9 months.

A comparison of the atomic composition of siderosomes (haemosiderin) in cryosections of unfixed frozen tissues and Epon-embedded tissues.

Siderosomes (i.e. single membrane-bound lysosomal bodies containing haemosiderin) were produced in the liver and muscle of rats by injections of iron dextran. Electron-probe X-ray analysis was executed on siderosomes in cryosections of quick-frozen fresh unfixed tissues (liver and muscle) and sections of Epon-embedded tissues. There were no statistically significant differences between the ratios of iron:phosphorus and iron:sulphur in these two types of preparations. Hence it is concluded that there is no significant loss or gain of phosphorus or sulphur during preparation of tissues for Epon embedding. The results confirm past belief that so little phosphorus or sulphur is present in siderosomes that haemosiderin is best regarded as ferric hydroxide oxide. A new finding in the present study was the demonstration of small amounts of potassium in siderosomes in cryosections. It seems that potassium is lost (leaches out) from siderosomes during preparation of tissues for Epon-embedding.

The morphology and atomic composition of aurosomes produced by sodium aurothiomalate in human monocytes.

Aurosomes were found in monocytes (from the peripheral blood of man) incubated with sodium aurothiomalate. In electron micrographs the aurosome presents as a single-membrane-bound lysosomal body with an electron-lucent or medium density matrix in which lie electron-dense filamentous (straight or curled), rod-like and lamellar profiles studded with particles and granules. Similar deposits did not develop in monocytes incubated with sodium thiomalate but myelinoid membranes and rod-like structures presumably derived from them were seen in the lysosomes of these cells. The aurosomes (and their characteristic electron-dense contents) produced in the monocytes in vitro were morphologically indistinguishable from those known to occur in various tissues of man and experimental animals treated with soluble goldsalts. The atomic composition of the aurosomes produced in vitro appears to be similar to that produced in vivo for in both instances Au, P and S can be demonstrated in the aurosome. It is concluded that the aurosomes produced in our experimental model is an accurate copy of that found in in vivo situations.

Ultrastructure of experimentally produced subcutaneous haematomas in the rabbit.

Haematomas were produced in rabbits by subcutaneous injection of autologous blood. Clotting and marked lysis of erythrocytes was noted in these haematomas, but there was no evidence of fragmentation of erythrocytes prior to or after ingestion by macrophages as has been reported in other sites such as the peritoneal cavity and the joint cavity. The phagocytosis of intact erythrocytes, lysed erythrocytes and haemoglobin led to the formation of three main types of lysosomal bodies; (1) myelinosomes containing whorled osmiophilic membranes, (2) siderosomes containing haemosiderin, and (3) myelinosiderosomes containing a mixture of osmiophilic membranes and haemosiderin.

Satellite heterophagosomes.

An incidental observation made during a recent ultrastructural study shows that after ingestion of an erythrocyte by a Kupffer cell surface budding occurred from the heterophagosome leading to the formation of smaller satellite or daughter heterophagosomes. It is suggested that an entire erythrocyte may be too large to digest in a single heterolysosome and that fragmentation into smaller units facilitates digestion by bringing about better contact between the enzymes and substrates.

Long term effects of myochrysine in articular cartilage.

Intra-articularly administered sodium aurothiomalate (Myochrysine) produced aurosomes containing characteristic electron dense contents (indicating the presence of gold), in the chondrocytes of rabbit articular cartilage. At first the aurosomes were bounded by a membrane but later the electron dense contents were seen lying free in the cytoplasmic matrix. Such deposits were detectable up to 14 months after injection of Myochrysine but none were found at later time intervals (18 months and 2 years). There was a reduction in the population of superficial chondrocytes (Zone I) while those in deeper zones (Zones II and III) showed an increased content of intracytoplasmic filaments. It is thought that these are regressive or degenerative changes produced by gold.

Genesis of concentric laminated inclusions in the nucleus.

Single-membrane-bound inclusions containing zymogen granules were found in pancreatic acinar cells. Images were seen suggesting that the concentric laminated inclusion is derived by hydration and fusion of such granules within the inclusion.

Intramatrical lipidic debris and calcified bodies in human semilunar cartilages.

Intramatrical lipid debris and calcified bodies were found in the matrix of injured and uninjured portions of semilunar cartilages obtained from patients ranging in age from 16 to 60 years. Most of the calcified bodies were homogeneously electron-dense and they were roughly spherical or cuboidal in shape. Much rarer was the occurrence of calcified bodies containing long, slender crystals. Electron-probe X-ray analysis showed that the homogeneously electron-dense bodies contain phosphorus, calcium and magnesium.

Electron-probe x-ray analysis of granules and particles found in aurosomes produced by colloidal gold.

Aurosomes produced in the rabbit synovial membrane after intraarticular injection of colloidal gold were found to contain large spherical electron-dense granules and fine electron-dense particles. Electron-probe x-ray analysis demonstrated the presence of gold in the granules and iron in the particles. Sulphur and phosphorus were not detected in these aurosomes produced by colloidal gold. This is in contrast to the aurosomes produced by the soluble gold salt sodium aurothiomalate where besides gold, sulphur and phosphorus are easily detected.

Ultrastructure of normal and torn menisci of the human knee joint.

Normal human menisci obtained at autopsy (seven cases) and the injured and uninjured portions of torn menisci obtained at surgery (nine cases) were studied with the electron microscope. The surface of menisci is composed of collagen fibrils surmounted by an electron-dense surface coat. Most of the cells in menisci are chondrocytes but a few fibroblasts and cells of an intermediate form difficult to classify as either fibroblasts or chondrocytes also occur. Mast cells are found at the vascularised periphery of the meniscus. Myofibroblasts were found in the injured portions of menisci in three out of the nine cases studied. A territorial matrix containing fibrils and proteoglycan particles with associated filaments is seen around or adjacent to chondrocytes, but sometimes this matrix is sparse or absent. The interterritorial or general matrix comprises collagen fibrils of widely varying diameters (25-180 nm) set in a sparse interfibrillary matrix containing proteoglycan particles. A few mature elastic fibres and several small or immature elastic fibres and collections of electron-dense filaments are seen in the general matrix. Also seen in this region are calcified bodies and matrical lipidic debris derived by the shedding of cell processes and in situ necrosis of cells. Other features seen in the matrix of the injured portion of the meniscus include: (1) membrane-bound cystic structures; (2) parting and fraying of collagen fibrils; and (3) pools of proteoglycan particles.

Myofibroblastoma: a tumour of myofibroblasts.

A myofibroblastoma occurring in the abdominal cavity of a 15 year old boy is described. This tumour was diagnosed as a low grade sarcoma by light microscopy but electron microscopy showed that the tumour was composed almost entirely of myofibroblasts and a few macrophages. Intermediate forms between myofibroblasts and macrophages were not seen nor were any fibroblasts seen in the main tumour mass. Total excision was impossible because the tumour had trapped loops of bowel and was adherent to the abdominal organs. The patient died of cachexia and haemorrhage but there were no distant metastases nor was there any marked infiltration of the abdominal organs. This case and a review of the literature shows that myofibroblastomas are locally aggressive tumours which do not metastasize and that if total excision is possible an uneventful recovery can be expected.